Primary hyperparathyroidism is characterized by reduced suppression, of PTH release by high Ca++ and increased parathyroid tissue mass. Low extracellular (EC) Ca++ maximally stimulates while high EC Ca++ inhibits PTH release. The regulation of PTH release by IC Ca++ is unusual. High EC Ca++ appears to suppress PTH release by increasing intracellular (IC) free Ca++. However, the IC mediators that transmit the hypocalcemic signal for maximal PTH release have not been identified. Phorbol esters which activate protein kinase C reverse the inhibitory effects of high EC Ca++ and stimulate PTH release. In other cells, polyphosphoinositide (PPI) turnover regulates secretion by generating two synergistic second-messengers -- diacylglycerol, an endogenous activator of protein kinase C, and inositol-1,4,5- triphosphate (IP3), a cellular Ca++-mobilizing compound. Thus, indirect studies suggest that PPI turnover in parathyroid cells activates two antagonistic pathways. We therefore propose to test the hypothesis that low Ca++-stimulated PTH release involves activation of C-kinase and that the generation of IP3 regulates the EC Ca++-induced rise in IC Ca++ thereby suppressing PTH release. To test this hypothesis, we will measure G-kinase activity and the breakdown products of PPI hydrolysis diacylglycerol and the inositol phosphates -- at different EC Ca++ concentrations. Activation of C-kinase will be assessed by 1) quantifying the translocation of Ca++-, phospholipid-dependent phosphotransferase activity from cytosolic to membrane fractions, and 2) comparing phosphorylation patterns from cells exposed to different EC Ca++, phorbol esters, and Ca++-ionophore. We will determine whether high EC Ca++ generates 1,4,5-IP3 and inositol-tetrakisphosphate (IP4), a proposed regulator of membrane Ca++ influx, and whether high EC Ca++ modifies the kinase responsible for producing IP4 from 1,4,5- IP3. We will then determine whether a GTP-binding protein couples changes in EC to increases in IC free Ca++. We will test this idea by measuring IC free Ca++ and PTH release after incorporation of GTP or a stable GTP analog into intact parathyroid cells or after pretreatment with pertussis or cholera toxins agents which ADP- ribosylate GTP-binding proteins and thus modify their activity. These studies should establish the role of PPI turnover and C- kinase in Ca++-regulated PTH release and may identify potential cellular defects involved in disordered regulation of PTH release by Ca++ characteristic of hyperparathyroidism.